New Natural Noncannabinoid Ligands for Cannabinoid Type-2 (CB2) Receptors

Since the discovery that Δ ⁹-tetrahydrocannabinol and related cannabinoids from Cannabis sativa L. act on specific physiological receptors in the human body and the subsequent elucidation of the mammalian endogenous cannabinoid system, no other natural product class has been reported to mimic the effects of cannabinoids. We recently found that N-alkyl amides from purple coneflower (Echinacea spp.) constitute a new class of cannabinomimetics, which specifically engage and activate the cannabinoid type-2 (CB₂) receptors. Cannabinoid type-1 (CB₁) and CB₂ receptors belong to the family of G protein-coupled receptors and are the primary targets of the endogenous cannabinoids N-arachidonoyl ethanolamine and 2-arachidonoyl glyerol. CB₂ receptors are believed to play an important role in distinct pathophysiological processes, including metabolic dysregulation, inflammation, pain, and bone loss. CB₂ receptors have, therefore, become of interest as new targets in drug discovery. This review focuses on N-alkyl amide secondary metabolites from plants and underscores that this group of compounds may provide novel lead structures for the development of CB₂-directed drugs.     

Transient over-expression of barley BAX Inhibitor-1 weakens oxidative defence and MLA12-mediated resistance to Blumeria graminis f.sp. hordei

BAX Inhibitor-1 (BI-1) is a conserved cell death suppressor protein. In barley, BI-1 ( HvBI-1 ) expression is induced upon powdery mildew infection and when over-expressed in epidermal cells of barley, HvBI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis . We co-expressed mammalian pro-apoptotic BAX together with HvBI-1, and the mammalian BAX antagonist BCL-X_L in barley epidermal cells. BAX expression led to cessation of cytoplasmic streaming and collapse of the cytoplasm while co-expression of HvBI-1 and BCL-X_L partially or completely, respectively, rescued cells from BAX lethality. When B. graminis was attacking epidermal cells, a green fluorescent protein fusion of HvBI-1 accumulated at the site of attempted penetration and was also present around haustoria. Over-expression of HvBI-1 in epidermal cells weakened a cell-wall-associated local hydrogen peroxide burst in a resistant mlo -mutant genotype and supported haustoria accommodation in race-specifically resistant MLA12 -barley. HvBI-1 is a cell death regulator protein of barley with the potential to suppress host defence reactions.     

Automatic Cell Counting in Continuous Flow Cultures of Tetrahymena pyriformis*

This report describes an electronic cell counter constructed for determining cell number in cultures of the ciliate, Tetrahymena pyriformis. The culture chamber has been equipped with a device which determines the number of cells per unit volume and records the number automatically. As cell multiplication is unaffected by the counting procedure the cells are returned to the culture. Furthermore, keeping the culture volume constant we have arranged a continuous flow of fresh nutrient medium through the culture chamber and thus established conditions under which cell multiplication has continued for months while determinations of cell concentrations have been recorded every 10 min. Since the culture volume has been small, ～25 ml, growth studies utilizing this method require less than one liter of fresh medium per week in spite of the fast multiplication (9 generations per 24 hr) occurring in cultures of Tetrahymena pyriformis under optimal conditions.     

Revision of the Genus Askenasia Blochmann, 1895, with Proposal of Two New Species, and Description of Rhabdoaskenasia minima N. G., N. Sp. (Ciliophora, Cyclotrichida)

ABSTRACT. The planktonic ciliate genus Askenasia Blochmann, 1895 is reviewed and the new genus Rhabdoaskenasia n. gen. is established. Askenasia is characterized by three circumferential kinety belts and a circumoral wreath of paired argyrophilic granules without recognizable cilia and nematodesmata. A "brush" is absent. Askenasia apparently lacks the key characters of the Haptorida and is thus transferred to the Cyclotrichida, family Mesodiniidae. Rhabdoaskenasia differs from Askenasia in having single files of basal bodies in all kinety belts and club-shaped extrusomes. It possesses a circumoral kinety composed of dikinetids from which nematodesmata originate, forming a distinct rhabdos. Although very similar to Askenasia in its general appearance, R. minima n. sp. could belong to another order. Based on an extensive review of the literature and on silver impregnated specimens the following Askenasia species are recognized and described in detail: A. volvox (Eichwald, 1852) Kahl, 1930, A. stellaris (Leegaard, 1920) Kahl, 1930, A. acrostomia n. sp., and A. chlorelligera n. sp. Askenasia faurei Kahl, 1930 and A. humilis Gajewskaja, 1928 are transferred to the genus Cyclotrichium: C. faurei (Kahl, 1930) n. comb., C. humilis (Gajewskaja, 1928) n. comb. The systematic position of the genus Askenasia is discussed and keys to the genera of the Mesodiniidae and to the species of Askenasia are provided.     

Unusual tubulins with deviant genetic code in the hypotrich ciliate Euplotidium itoi

Recently, we have shown the presence of molecules belonging to the cholinergic signalling system in the ciliate Paramecium primaurelia and in the sarcodine Dictyostelium discoideum . Propionylcholinesterase (PrChE) activity has been detected in single-cell amoebae of D. discoideum , using cytochemical, electrophoretic, and spectrophotometric methods (Falugi et al., Chemosphere , 48:407–414, 2002; Amaroli et al., Europ. J. Protistol ., 39:213–222, 2003). It has been suggested that this enzyme activity is involved in cell–cell and cell–environment interactions, as its inhibition by xenobiotic compounds affects cell migration and aggregation. In this work, we have spectrophotometrically evaluated the effect of an ELF-EMF of 300 T, 50 Hz, on PrChE activity after exposure of single-cell amoebae from 1 h up to 48 h at 22°C. The enzyme activity was inhibited significantly by 1-h- and 3-h exposures, whereas it was similar to the control value after a 4-h exposure. A significant increase in PrChE activity was found after 5- and 24-h exposures, while a decrease in PrChE activity appeared in 48-h-exposed samples. The increased PrChE activity detected in 24-h-exposed cells returned to the control value 24 h after transferring the amoebae to standard conditions. A delay in both migration and aggregation processes was observed in 3 h-exposed cells, corresponding to a decreased PrChE activity. After a 24-h exposure, a decrease in the fission rate and an increase in the cell size were found.     

The effect of ozone on the emission of carbonyls from leaves of adult Fagus sylvatica

Under the site conditions of a temperate forest, the exchange of short-chained oxygenated carbonyls (aldehydes, ketones) was assessed from leaves of adult European beech trees. The crowns of the trees were either exposed to an elevated O₃ regime as released by a free-air fumigation system (2 × O₃) or to the unchanged O₃ regime at the site (1 × O₃, ‘control’). Daily fluctuations of oxygenated carbonyls were quantified in relation to environmental and physiological factors. In particular, the effect of O₃ on carbonyl exchange was studied. Measurements of leaf gas exchange were performed with a dynamic cuvette system, and carbonyl fluxes were determined using 2,4-dinitrophenylhydrazine (DNPH)-coated silica gel cartridges. Leaves mainly emitted acetaldehyde, formaldehyde and acetone. Acetaldehyde dominated the emissions, amounting up to 100 nmol m⁻² min⁻¹, followed by formaldehyde (approximately 80 nmol m⁻² min⁻¹) and acetone (approximately 60 nmol m⁻² min⁻¹). Carbonyl emissions were highest during midday and significantly lowered at night, irrespective of the O₃ exposure regime. Trees exposed to 2 × O₃ emitted acetaldehyde and acetone at enhanced rates. The findings are of particular significance for future climate change scenarios that assume increased O₃ levels.     

Pair bond and breeding success in Blue Tits Parus caeruleus and Great Tits Parus major

Data from 939 nests of the Blue Tit Parus caeruleus and 1008 nests of the Great Tit P. major from nestboxes provided in superabundance in mixed forest study sites between 1976 and 2001 were analysed to examine the effects of mate retention on breeding success and the relationship between mate fidelity and site fidelity. Most birds retained their former partner (76% in Great Tits and 65% in Blue Tits). The probability of a pair divorcing was affected by male age in Great Tits, divorce being more likely in pairs with first‐year males. Great Tit pairs breeding together for a second season bred earlier, but had no higher breeding success than pairs breeding together for the first time. In Blue Tits laying date and start of incubation tended to be earlier in pairs breeding together for a second season, but hatching and fledging dates were not earlier than in other pairs. Great Tit pairs breeding together for two consecutive seasons bred earlier in the second season than in the first, but breeding success did not differ significantly between years. In both species, breeding performance did not differ between pairs that divorced after a season and pairs that stayed together. Thus breeding success did not determine whether a pair divorced or bred together again. Neither Blue Tits nor Great Tits improved their breeding performance through divorce. Blue Tit females even had fewer fledglings in the year after divorce than in the year before. Mate retention affected breeding site fidelity. Blue Tit females had greater breeding dispersal distances between consecutive years when re‐mating than when breeding again with the same mate. In Great Tits both males and females dispersed more when re‐mating than when retaining the former partner, suggesting that mate retention increased the chance of retaining the breeding site. In both species, breeding dispersal distances did not differ between pairs that divorced and pairs in which one mate disappeared. Because no major advantage of mate retention was evident, we suggest that mate retention evolved under different conditions than those found in study sites with high breeding densities and a superabundance of artificial nesting sites.     

A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato

Genes for seven putative serine proteases (ChpA–ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis ( Cmm ) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm . The genes chpA , chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.